The development of peptide- or polypeptide-based drug candidates often starts with the screening of libraries of related peptide or polypeptide sequences. Thus the first step for the selection of therapeutic antibody candidates usually is the creation of a highly diverse library of antibody sequences.
Several methods for the design and construction of diverse antibody libraries are known in the art.
It has been described that the diversity of a filamentous phage-based combinatorial antibody library can be increased by shuffling of the heavy and light chain genes (Kang et al., Proc. Natl. Acad. Sci. USA, 88:11120-11123, (1991)) or by introducing random mutations into the library by error-prone polymerase chain reactions (PCR) (Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580, (1992)). The use of defined frameworks as the basis for generating antibody libraries has been described by Barbas et al., Proc. Natl. Acad. Sci. USA 89:4457-4461 (1992) (randomizing CD3-H3); Barbas et al., Gene 137:57-62 (2003) (extending randomization to Vκ CDR3); and Hayanashi et al., Biotechniques 17:310 (1994) (simultaneous mutagenesis of antibody CDR regions by overlap extension and PCR). Others report combination of CDR-H3 libraries with a single VL gene (Nissim et al., EMBO J. 13:692-698 (1994)), a limited set of VL genes (De Kruif et al., J. Mol. Biol. 248:97-105 (1995)); or a randomized repertoire of VL genes (Griffiths et al., EMBO J. 13:3245-3260 (1994)).
See also U.S. Pat. Nos. 5,667,988; 6,096,551; 7,067,284 describing methods for producing antibody libraries using universal or randomized immunoglobulin light chains.
Knappik et al., J. Mol. Biol. 296:57-86 (2000) describe a different concept for designing and constructing human antibody libraries, designated HuCAL (Human Combinatorial Antibody Libraries). This approach is based on the finding that each of the human VH and VL subfamilies that is frequently used during an immune response is represented by one consensus framework, resulting in seven HuCAL consensus genes for heavy chains and seven HuCAL consensus genes for light chains, which yield 49 possible combinations. All genes are made by total synthesis, taking into consideration codon usage, unfavorable residues that promote protein aggregation, and unique and general restriction sites flanking all CDRs. The approach leads to the generation of modular antibody genes containing CDRs that can be converted into different antibody formats, as needed. The design and synthesis of HuCAL antibody libraries is described in U.S. Pat. Nos. 6,300,064; 6,696,248; 6,706,484; and 6,828,422.
Despite these and other advances there a great need for new, efficient methods for the design and construction of highly diverse (poly)peptide, such as antibody, libraries.